Microbial agent and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a microbial agent and application thereof. The active ingredient of the microbial agent is bacillus velezensis, the bacillus velezensis is bacillus velezensis XM18-5, the bacillus velezensis is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC 25698. The microbial agent provided by the invention can effectively inhibit various pathogenic bacteria such as potato early blight, late blight and the like and promote plant root growth and emergence rate at the same time, and the formula is environment-friendly and suitable for green agricultural production.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a microbial agent and application thereof. Background Art

[0002] As one of the world's most important food crops, potatoes are often attacked by pests during their growth, resulting in serious yield losses. Although traditional chemical pesticides have certain control effects, long-term use can easily lead to problems such as pathogen resistance, environmental pollution, and soil microecological imbalance. In recent years, microbial agents have become a research hotspot due to their environmental friendliness and sustainability. Among them, Bacillus has attracted much attention in the field of biocontrol due to its strong spore production ability and high stress resistance. However, existing microbial agents generally have problems such as unstable strain activity, insufficient formula adaptability, and a single control target, which limits their practical application effect.

[0003] Although Bacillus velezensis has been reported to have antibacterial potential, the development of specific strains and their corresponding formulations for potato blight is still relatively scarce. Furthermore, the selection and proportions of components (such as carriers, dispersants, and preservatives) in existing microbial formulations often lack scientific optimization, resulting in low bacterial powder activity preservation and insufficient colonization ability after field application. Regarding preparation processes, liquid fermentation, while widely used, has drawbacks such as high cost and low bacterial cell concentration. Further research is needed into solid-state fermentation technology using agricultural waste as a substrate to improve bacterial powder yield and functional stability.

[0004] At the same time, existing microbial agents are primarily focused on single disease control, with insufficient research on their combined effects on enhancing potato emergence and promoting growth. Therefore, there is an urgent need to develop a highly effective, multifunctional microbial agent tailored to potato needs. Through formulation optimization and process improvements, this could synergize disease control with crop growth. Summary of the Invention

[0005] The present invention aims to provide a microbial agent based on the active ingredient of Bacillus velezensis XM18-5, which can effectively inhibit various pathogens such as potato early blight and late blight, while promoting plant root growth and seedling emergence rate, achieving synergistic promotion of disease prevention and control and crop growth. The formula is environmentally friendly and suitable for green agricultural production.

[0006] To achieve the above objectives, on the one hand, the present invention provides a microbial agent, the active ingredient of the microbial agent is Bacillus velezensis XM18-5, and the Bacillus velezensis is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC 25698.

[0007] The above-mentioned microbial agent is composed of the following components in percentage by mass: 30% to 40% carrier, 2% to 8% wetting agent, 8% to 16% dispersant, 0.2% to 0.8% protective agent, and 35.2% to 59.8% bacterial powder;

[0008] The carrier is one of diatomaceous earth, kaolin, attapulgite, and white carbon black; the wetting agent is one of sodium lauryl sulfate, polyethylene glycol, and calcium chloride; the dispersant is one of sodium lignosulfonate, sodium tripolyphosphate, sodium methylene disulfonate, gum arabic, and sodium carboxymethyl cellulose; and the protective agent is one of sodium humate, xanthan gum, kaolin, dextrin, and soluble starch;

[0009] The bacterial powder includes Bacillus velezensis XM18-5.

[0010] The above-mentioned microbial agent is composed of the following components in percentage by mass: 35% diatomaceous earth, 8% polyethylene glycol, 12% sodium carboxymethyl cellulose, 0.8% soluble starch, and 35.2% bacterial powder.

[0011] The above-mentioned microbial agent, calculated by mass percentage, the preparation steps of the bacterial powder include: calculated by mass percentage, the preparation steps of the bacterial powder include:

[0012] Step 1: Seed solution preparation:

[0013] Step 101: The strain XM18-5 was transferred to PDA medium and activated for 24 hours. A single colony was picked and streaked for subculture, and cultured for two generations until the normal metabolic level was reached.

[0014] Step 102: Pick a ring of XM18-5 and transfer it to NB medium, culture it at 30°C and 180 rpm for 24 hours to prepare the seed solution for later use;

[0015] Step 2: Fermentation broth preparation:

[0016] Step 201: Inoculate the reserved seed solution in step 102 into NB culture medium at an inoculum volume of 5% to 10% (V / V), and culture at 34° C. and 180 rpm for 48 hours;

[0017] Step 202: Measure the spore rate using a hemocytometer to ensure that the spore rate is ≥90%, and the fermentation liquid is ready for use.

[0018] Step 3: Centrifuge the fermentation broth at 4°C and 8000 rpm for 10 min, discard the supernatant, and obtain a concentrated bacterial solution;

[0019] Step 4: Preparation of bacterial powder:

[0020] Step 401: mixing 60% to 65% corn straw, 20% to 30% bran, 2.5% to 3.0% corn, 1.40% to 1.48% ammonium sulfate, and 0.10% to 0.15% manganese sulfate, adding 6% to 10% of the concentrated bacterial solution obtained in step 3 as an inoculum, and solid-state fermenting in a 37° C. incubator for 48 hours to obtain a fermentation product;

[0021] Step 402: drying the fermentation product obtained in step 401 in a 45° C. oven and then ultrafine grinding the product to prepare final bacterial powder.

[0022] On the other hand, the present invention also provides a microbial granule, which is prepared by adding a binder to the above-mentioned microbial agent.

[0023] On the other hand, the present invention also provides the use of the above-mentioned microbial agent or microbial granule in the prevention and treatment of potato blight.

[0024] In the above application, the diseases include potato early blight, potato late blight, potato black mole, potato scab and potato dry rot.

[0025] On the other hand, the present invention also provides the use of the above-mentioned microbial agent or the above-mentioned microbial granule in improving the potato emergence rate.

[0026] On the other hand, the present invention also provides the use of the above-mentioned microbial agent or the above-mentioned microbial granule in promoting potato growth.

[0027] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0028] 1. The microbial agent of the present invention can achieve broad-spectrum antibacterial and disease prevention and control; the microbial agent of the present invention is based on the lipopeptide antibacterial substance secreted by Bacillus Velez-Bacillus XM18-5, which destroys the integrity of the pathogen cell membrane, inhibits the activity of key enzymes, and interferes with mycelial growth. It achieves broad-spectrum antagonism against potato diseases such as early blight and late blight, while reducing the risk of pathogen resistance.

[0029] 2. The present invention optimizes the culture medium formula to achieve synergistic efficiency; adopts a solid-state fermentation process using corn straw as a carbon source, utilizes the microorganism's ability to degrade cellulose to promote bacterial spore production, combines diatomaceous earth carriers with soluble starch protective agents to form a hydrophobic barrier to resist environmental stress, and significantly improves the activity and storage stability of bacterial powder.

[0030] 3. The microbial agent provided by the present invention achieves plant growth promotion and systemic resistance induction; the indoleacetic acid and other plant hormones produced by the metabolism of strain XM18-5 (Bacillus Velez XM18-5) directly stimulate root development, and at the same time enhance systemic resistance by activating the plant jasmonic acid signaling pathway, synergistically improving the emergence rate and stress resistance, and realizing dual regulation of disease prevention and control and crop growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 These are morphological images of Bacillus velezensis XM18-5; a is the colony morphology, b is the Gram staining microscopy result, and c is the electron microscope scanning image.

[0033] Figure 2 This is the 16S rDNA phylogenetic tree of Bacillus velez XM18-5.

[0034] Figure 3 This is the gyrB phylogenetic tree of Bacillus velez XM18-5.

[0035] Figure 4 This is the growth curve of Bacillus velez XM18-5.

[0036] Figure 5 This is a bar graph showing the effect of carbon source on shake flask fermentation of strain XM18-5.

[0037] Figure 6 This is a bar graph showing the effect of nitrogen source on shake flask fermentation of strain XM18-5.

[0038] Figure 7 This is a bar graph showing the effect of inorganic salts on shake flask fermentation of strain XM18-5.

[0039] Figure 8 This is the optimization discount diagram of the fermentation conditions of strain XM18-5; where A is pH; B is the inoculum size; C is the culture temperature; D is the liquid volume; E is the rotation speed; and F is the culture time.

[0040] Figure 9 FIG is a wettable powder prepared according to the present invention.

[0041] Figure 10 This is a diagram of the microbial granules prepared according to the present invention.

[0042] Figure 11 This is a diagram illustrating the aboveground and underground biomass and growth of individual plants under different treatments.

[0043] Figure 12 This is a diagram illustrating the aboveground and underground biomass and growth of individual plants under different treatments.

[0044] Figure 13 The bar graphs show the plant heights of each treatment at different periods.

[0045] Figure 14 The bar graphs show the root lengths of each treatment at different periods.

[0046] Figure 15 The bar graphs show the stem thickness of each treatment at different periods. DETAILED DESCRIPTION

[0047] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental and detection methods described in each example are conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified. The ratios in the following examples are ratios by mass unless otherwise specified.

[0048] Example 1

[0049] This example provides the isolation and identification results of Bacillus velezinis XM18-5 (antagonistic strain).

[0050] 1. Isolation of antagonistic strains:

[0051] Isolation of strains: Soil dilution separation method was used for separation. After drying the soil sample, weigh 1g and add 9mL sterile water to make 10 -4 , 10 -5 , 10 -6 and 10 -7 Take 100 μL of the dilution solution and spread it on the OMA solid culture medium plate. Repeat each gradient 3 times. After culturing in the dark at 30℃ for 18-24h, pick out single colonies with different morphologies and streak to separate them to obtain pure culture. Transfer them into the slant of freezing tubes for later use.

[0052] 2. Identification of morphological and physiological and biochemical characteristics of antagonistic strains:

[0053] The isolated antagonistic strain was streaked onto LB solid medium, and the colony morphology was observed and identified. Physiological and biochemical indicators such as VP, glucose glycolysis, starch hydrolysis, gelatin liquefaction, and nitrate reduction of strain XM18-5 were identified using HBI Bacillus bioassay strips.

[0054] The morphological identification results are as follows Figure 1 As shown in a, strain XM18-5 grows well on LB solid medium. The single colony is milky white, spherical, opaque, slightly convex, with slightly wrinkled surface, irregular edges, and no pigmentation around it. Figure 1 As shown in Figure b, the strain was found to be Gram-positive under microscopic examination. Figure 1 As shown in Figure (c), scanning electron microscopy results show that the XM18-5 bacteria are rod-shaped, with a smooth surface and no wrinkles. Based on morphological identification, it is preliminarily determined that the strain XM18-5 belongs to the genus Bacillus.

[0055] Physiological and biochemical characterization results, as shown in Table 1, show that strain XM18-5 exhibits catalase and oxidase activities, can utilize citrate, propionate, D-xylose, L-arabinose, glucose, and sucrose, and is positive for VP and nitrate reduction reactions. It grows normally in media with a pH of 5.7 and a 7% NaCl concentration, respectively. It can hydrolyze starch and gelatin, but cannot utilize D-mannitol, maltose, or lactose. It produces urease but not esterase. Based on these physiological and biochemical tests, combined with morphological characteristics, it was preliminarily identified as Bacillus velezensis.

[0056] Table 1: Physiological and biochemical characteristics of strain XM18-5

[0057] Test indicators result Catalase + Oxidase + VP + Citrate + Propionate + D-Xylose + L-arabinose + D-Mannitol - Gelatin liquefaction + 7% NaCl + Growth at pH 5.7 + Nitrate reduction + starch hydrolysis + maltose - glucose + sucrose + lactose - urease + Esterase -

[0058] Note: +: positive; -: negative.

[0059] 3. Molecular biological identification of antagonistic strains:

[0060] The genomic DNA of strain XM18-5 was extracted according to the instructions of the Solarbio Bacterial Genomic Kit (Cat#D1600), and the 16S rDNA sequence was amplified using bacterial universal primers using this as a template;

[0061] The bacterial universal primers are specifically:

[0062] 27F(5′-AGTTTGATCMTGGCTCAG-3′);

[0063] 1492R(5′-GGTTACCTTGTTACGACTT-3′).

[0064] In this example, the sequence of the gyrase β subunit gene (gyrB) was amplified using bacterial housekeeping gene primers.

[0065] The bacterial housekeeping gene primers are specifically:

[0066] gyrB-F(5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYG A-3′);

[0067] gyrB-R(5′-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCN GTCAT-3′).

[0068] The above primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the PCR amplification products were sequenced by Shanghai Sangon Biotechnology Co., Ltd. The sequencing results were uploaded to the NCBI website and compared with the 16S rDNA and gyrB gene sequences of other strains by BLAST. The strain sequences with high similarity in GenBank were selected, and a phylogenetic tree was constructed using the neighbor-joining method using MEGA.11 software.

[0069] Based on 16S rDNA gene sequencing, a 1446 bp sequence was obtained and registered at NCBI with the accession number OK560566. BLAST comparison revealed that strain XM18-5 had a sequence similarity of greater than 98% with several Bacillus strains, confirming that strain XM18-5 belongs to the genus Bacillus. Download the 16S rDNA sequence of a strain with high sequence homology to the target strain and construct the following: Figure 2 The phylogenetic tree of strain XM18-5 is shown. The results show that strain XM18-5 and Bacillus velezensis BC RC 17467 are clustered on the same root branch with a similarity of 100%, indicating that strain XM18-5 has a high homology with Bacillus velezensis and can be preliminarily determined to be Bacillus velezensis.

[0070] Based on the sequencing of gyrB gene, a sequence of 1137 bp was obtained, which was registered at NCBI and obtained the sequence accession number OK557802. Figure 3 The phylogenetic tree of the strains shown in the figure shows that strain XM18-5 and Bacillus velezensis BCRC 17467 clustered into a branch with a sequence similarity of 100%. Combined with the 16S rDNA gene sequencing results, it was shown that the biocontrol strain XM18-5 was Bacillus velezensis.

[0071] The deposit information of Bacillus velez XM18-5 is as follows:

[0072] Strain name: XM18-5;

[0073] Classification name: Bacillus velezensis;

[0074] Date of receipt by the depository: September 13, 2022;

[0075] Time of issuance of the preservation certificate: September 30, 2022;

[0076] Depository: General Microbiology Center, China Culture Collection Administration;

[0077] Deposit number: CGMCC 25698;

[0078] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0079] Example 2

[0080] This example is intended to illustrate the optimization of fermentation medium components for Bacillus velez XM18-5 and to demonstrate the optimal fermentation conditions for strain XM18-5.

[0081] 1. Growth curve determination of strain XM18-5.

[0082] Remove the Bacillus velezensis XM18-5 strain from the refrigerator and culture it in LB liquid medium. After 12 hours, inoculate the colony onto LB solid medium using the streak plate method or the dilution spread plate method. After 24 hours, wait for a single colony to grow, pick a single colony and inoculate it into LB liquid medium. Culture it at 28°C and 180 rpm for 24 hours to prepare a bacterial suspension. Then, inoculate the bacterial suspension into a new LB liquid medium at a 5% inoculum size and culture it with shaking. Measure the OD value within a certain period of time. 600 200 μL of bacterial suspension was added to a 96-well plate, and the absorbance was measured at 600 nm using a microplate reader. LB medium was used as a blank. Three replicates were taken each time to reduce experimental errors. Growth curves were prepared using GraphPad Prism 8.0.

[0083] The strain XM18-5 was grown with the OD value at 600nm as the ordinate and the culture time as the abscissa (0-48h). The absorbance value at 600nm was measured every 2 hours to draw a standard growth curve of the target strain. The results are as follows: Figure 4 The results showed that under the culture conditions provided by the laboratory, the strain XM18-5 grew slowly between 0 and 4 hours, and entered the logarithmic growth phase between 4 and 30 hours. The bacterial metabolism was vigorous and the reproduction rate was fast. The stable period was 30 to 36 hours, and the OD at 34 hours was 0. 600 The value reaches the maximum, at this time the number of microorganisms is the largest and is in a stable period, and after 34 hours the bacteria gradually die, OD600 Therefore, the subsequent test research cycle was set at 34h to ensure the accuracy of the test results.

[0084] 2. Screening of fermentation medium components.

[0085] The carbon source culture medium was an equal amount (5.0 g / L) of maltose, lactose, glucose, sucrose, corn flour and soluble starch instead of the carbon source yeast powder in the initial culture medium. The nitrogen source (peptone 10.0 g / L) and inorganic salt (NaCl 10.0 g / L) remained unchanged. The culture conditions were 250 mL triangular flask with a liquid volume of 100 mL, 2% seed liquid inoculation, 30 ° C, 180 r / min, shaking culture for 24 h, and the OD of the fermentation reaction liquid was measured. 600 value, repeated 3 times, and the optimal carbon source was screened out; the nitrogen source culture medium was an equal amount (10.0 g / L) of peptone, urea, tryptone, L-asparagine, yeast extract and beef extract instead of the nitrogen source peptone in the initial culture medium, the carbon source was the optimal carbon source that had been screened, the inorganic salt (NaCl 10.0 g / L) remained unchanged, and the fermentation conditions were consistent with the carbon source to screen the optimal nitrogen source; the inorganic salt culture medium was an equal amount (10.0 g / L) of potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), magnesium sulfate (MgSO4), zinc sulfate (ZnSO4), calcium chloride (CaCl2) and sodium chloride (NaCl) to replace the inorganic salt (NaCl) in the initial culture medium, the carbon source and nitrogen source were the optimal components screened, the fermentation conditions remained unchanged to screen out the optimal inorganic salt, and the graph was drawn using GraphPadPrism9.3.2.

[0086] Based on the screening of single carbon source factors, Figure 5 It can be seen that different types of carbon sources have a certain effect on the number of viable bacteria of strain XM18-5. The results show that the culture medium with soluble starch as the carbon source can make strain XM18-5 obtain the maximum absorbance value, the highest bacterial concentration in the fermentation broth, and the OD 600 It reached 0.911, therefore, soluble starch was used as the optimal carbon source for XM18-5.

[0087] In the single factor screening test of nitrogen sources, six ingredients including peptone, urea, trypsin, L-asparagine, yeast extract and beef extract were selected as different nitrogen sources. Figure 6 As shown in the figure, different types of nitrogen sources have a certain effect on the viable bacterial count of strain XM18-5. When yeast extract is used as the only nitrogen source, the OD 600 The value is the largest, which is 1.226, followed by beef extract, so yeast extract is selected as the preferred nitrogen source for XM18-5 fermentation culture.

[0088] like Figure 7As shown in the figure, in the optimization experiment of single inorganic salt factor, KH2PO4, K2HPO4, MgSO4, ZnSO4, CaCl2 and NaCl were used as the only inorganic salts for single factor screening of inorganic salts. The screening results showed that different types of inorganic salts had a certain effect on the viable bacterial count of XM18-5. When K2HPO4 was used as the only inorganic salt, the OD value of bacterial solution increased. 600 The value is the largest, reaching 1.220, so K2HPO4 is selected as the optimal inorganic salt.

[0089] 3. Orthogonal experiment of fermentation medium components.

[0090] Based on the results of the single factor experiment, OD 600 The value is the indicator, according to L(16)4 3 Orthogonal array design with three factors and four levels was used to screen the optimal culture medium ratio.

[0091] According to the results of the single factor test of the culture medium, the best carbon source was soluble starch (factor A), and four levels were set at 5.00 g / L, 7.50 g / L, 10.00 g / L, and 12.50 g / L; the best nitrogen source was yeast extract (factor B), and four levels were set at 5.00 g / L, 7.50 g / L, 10.00 g / L, and 12.50 g / L; the best inorganic salt was K2HPO4 (factor C), and four levels were set at 5.00 g / L, 7.50 g / L, 10.00 g / L, and 12.50 g / L. A three-factor four-level orthogonal experiment was designed, and the OD value was used to determine the optimal carbon source. 600 The value is the optimization index and the optimal culture medium is selected. As shown in Table 2, the three factors have an impact on the OD of Bacillus velez XM18-5 shake flask fermentation. 600 The order of influence of the values ​​is B>C>A, that is, yeast extract concentration>K2HPO4 concentration>soluble starch concentration. When the combination is A4B4C1, the OD value of the shake flask fermentation of Bacillus velez XM18-5 is 600 The value reaches a maximum value of 1.37, that is, the concentration of soluble starch is 12.50 g / L, the concentration of yeast extract is 12.50 g / L, and the concentration of K2HPO4 is 5.00 g / L.

[0092] Table 2: Results of orthogonal experiment for culture medium optimization

[0093]

[0094]

[0095] Note: Data are means (n=3). SPSS 19.0 software was used to perform Duncan's multiple range test and one-way analysis of variance. Different letters indicate significant differences among treatments (p<0.05).

[0096] 4. Optimization of fermentation conditions of strain XM18-5.

[0097] Based on the optimized fermentation medium formula, the initial fermentation conditions of the medium are pH 7.0; temperature 28° C.; inoculation amount 2%; rotation speed 180 r / min; liquid volume 100 mL; and dark shaking culture for 24 h. The initial pH of the culture medium (6, 6.5, 7, 7.5, 8, 8.5); culture temperature (25℃, 27.5℃, 30℃, 32.5℃, 35℃, 37.5℃); inoculum size (2%, 4%, 6%, 8%, 10%, 12%, 14%); culture time (8h, 16h, 24h, 32h, 40h, 48h); rotation speed (120r / min, 140r / min, 160r / min, 180r / min, 200r / min, 220r / min); and liquid volume (50mL, 75mL, 100mL, 125mL, 150mL, 175mL) were changed respectively, and the OD of the fermentation broth obtained by the XM18-5 strain under different culture conditions was measured. 600 The values ​​were measured three times and the average value was taken. The obtained data were analyzed using GraphPad Prism 8.0 software.

[0098] After optimizing the fermentation conditions of strain XM18-5, including initial pH, inoculation size, culture temperature and liquid volume, the OD of strain XM18-5 after 24 h of constant temperature culture at 30℃ under different single factor conditions was determined. 600 Absorbance value. The results show that when other conditions are kept constant, such as Figure 8 As shown in A, when the culture pH is adjusted to 7.5, the absorbance is the highest, and OD 600 is 1.25; Figure 8 As shown in B, when the inoculation amount was 11%, the absorbance value was significantly higher than that of other treatments, and OD 600 The value is 0.74; Figure 8 As shown in C, when the culture temperature is set to 32.5℃, the number of viable bacteria reaches its peak, OD 600 The value is 1.43; Figure 8 As shown in D, when the liquid volume is 75mL / 250mL, OD 600 is 0.76; Figure 8 As shown in E, when the incubator speed is set to 160 rpm / min, OD 600 It was significantly higher than other treatments, which was 1.432. When the above optimal conditions were kept fixed, the strain XM18-5 was cultured under shaking conditions, and the absorbance value was measured every 8 hours. The growth curve was drawn according to the measurement results, as shown in the figure. Figure 8As shown in F, 8 to 24 hours is the logarithmic growth period of the strain, at which the growth rate is the fastest, 24 to 32 hours is the slow growth period, and the effective concentration of strain XM18-5 is the highest when the culture time is 32 hours. 600 The value is 1.294. When the culture time exceeds 40 hours, the strain enters the decline stage and the bacterial liquid concentration shows a downward trend.

[0099] Example 3

[0100] This example is intended to illustrate the component screening of the microbial agent provided by the present invention, wherein the microbial agent contains Bacillus Velezii XM18-5.

[0101] In this embodiment, the preparation of bacterial powder is first described:

[0102] Calculated by mass percentage, the preparation steps of the bacterial powder include:

[0103] Step 1: Seed solution preparation:

[0104] Step 101: The strain XM18-5 was transferred to PDA medium and activated for 24 hours. A single colony was picked and streaked for subculture, and cultured for two generations until the normal metabolic level was reached.

[0105] Step 102: Pick a ring of XM18-5 and transfer it to NB medium, culture it at 30°C and 180 rpm for 24 hours to prepare the seed solution for later use;

[0106] Step 2: Fermentation broth preparation:

[0107] Step 201: The reserved seed solution in step 102 is inoculated into NB medium at an inoculum volume of 8% (V / V), and cultured at 34° C. and 180 rpm for 48 h;

[0108] Step 202: Measure the spore rate using a hemocytometer. If the spore rate is ≥90%, the fermentation liquid is ready for use.

[0109] Step 3: Centrifuge the fermentation broth at 4°C and 8000 rpm for 10 min, discard the supernatant, and obtain a concentrated bacterial solution;

[0110] Step 4: Preparation of bacterial powder:

[0111] Step 401: mixing 60% corn straw, 25% bran, 2.5% corn, 1.40% ammonium sulfate, and 0.10% manganese sulfate, adding 10% of the concentrated bacterial solution obtained in step 3 as an inoculum, and solid-state fermenting in a 37° C. incubator for 48 hours to obtain a fermentation product;

[0112] Step 402: drying the fermentation product obtained in step 401 in a 45° C. oven and then ultrafine grinding the product to prepare final bacterial powder.

[0113] The live bacterial count of the bacterial powder reached up to 62 billion / g, and the spore formation rate reached up to 90%.

[0114] Activation of pathogens: Inoculate Streptomyces scabies X-1 onto new PDA culture medium and culture in a constant temperature incubator at 28°C for 72 hours.

[0115] 1. Screening of vectors.

[0116] Preparation of mother powder: The bacterial powder was mixed evenly with four carriers (diatomaceous earth, kaolin, attapulgite, and white carbon) in a ratio of 3:2. After ultrafine grinding, it was sieved (200 mesh) for later use. Three replicates were set for each sample, and the number of viable bacteria, wetting time, and suspension rate were measured.

[0117] Table 3 shows that samples using attapulgite and diatomaceous earth as carriers had higher viable bacterial counts, at 52.5 billion / g and 57.7 billion / g, respectively, with a small difference between the two. The wetting time and suspension rate of diatomaceous earth and kaolin were 47.67 seconds and 50.65%, respectively, and 82.77 seconds and 78.5%, respectively. Wettability is a key performance indicator for wettable powders of pesticides. A high suspension rate ensures that the inoculum is evenly suspended in water and evenly sprayed from the spray nozzle, achieving optimal control effectiveness. Furthermore, the practicality of the formulation should be considered, with a carrier that is low-cost, has abundant raw materials, and is easily transportable. Taking all these factors into consideration, diatomaceous earth was selected as the optimal carrier.

[0118] Table 3: Screening of vectors

[0119] carrier Number of viable bacteria / (100 million / g) Suspension rate / % Wetting time / s Kaolin 505.00±0.10b 78.50±0.58a 82.77±0.15d diatomite 577.00±0.07a 50.65±0.58d 47.67±0.33e Silica 385.50±0.19c 64.350±0.58b 146.67±0.33a Attapulgite 525.00±0.20b 44.840±0.58e 97.67±0.33b CK 493.00±0.18b 60.20±0.58c 84.67±0.33c

[0120] 2. Screening of wetting agents.

[0121] The wetting agents and mother powders were mixed evenly in a ratio of 1:5, and then ultrafinely ground and sieved (200 mesh) for later use. Three replicates were set for each sample to measure the number of viable bacteria, wetting time, and suspension rate.

[0122] Table 4 shows that the sample using polyethylene glycol as a wetting agent had the highest viable bacterial count, 60.53 billion / g. SDS and polyethylene glycol also had the highest suspension rates, at 75.69% and 77.57%, respectively. Polyethylene glycol had the shortest wetting time, at 50.09 seconds. Wetting agents can reduce the surface tension of water, facilitating the wetting of active ingredients by water and enhancing the efficacy of wettable powders. Therefore, when selecting wetting agents, their suspension rate and wettability should be considered. Taking all these factors into consideration, polyethylene glycol was selected as the optimal wetting agent.

[0123] Table 4: Screening of wetting agents

[0124]

[0125]

[0126] 3. Screening of dispersants.

[0127] Each dispersant and mother powder were mixed uniformly in a ratio of 1:4, ultrafinely ground, and then sieved (200 mesh) to prepare samples. Three replicates were set for each sample, and the number of viable bacteria, wetting time, and suspension rate were measured.

[0128] Table 5 shows that the sample using sodium carboxymethyl cellulose as the dispersant had the highest viable bacterial count and suspension rate, at 57.9 billion / g and 78.57%, respectively. The wetting time was ranked in the order of sodium methylene disulfonate > control > gum arabic > sodium carboxymethyl cellulose > sodium lignosulfonate > sodium tripolyphosphate. Dispersants can reduce the surface tension of bacterial powder, allowing for uniform dispersion in the medium and facilitating the stability of wettable powders. Sodium carboxymethyl cellulose achieved high viable bacterial counts, a high suspension rate, excellent wetting properties, and relatively low cost. Taking all factors into consideration, sodium carboxymethyl cellulose was selected as the optimal dispersant.

[0129] Table 5: Screening of dispersants

[0130] dispersants Number of viable bacteria / (100 million / g) Suspension rate / % Wetting time / s Sodium lignosulfonate 461.00±0.15c 71.35±0.06d 30.62±0.34d Sodium tripolyphosphate 395.00±0.16d 75.55±0.03b 23.35±0.69e Sodium methylene disulfonate 483.00±0.12c 73.32±0.06c 177.20±0.48a Gum Arabic 556.00±0.09ab 72.90±0.06c 79.31±0.43c Sodium carboxymethyl cellulose 579.00±0.04a 78.57±0.39a 48.66±0.88c CK 531.00±0.06b 59.55±0.17e 79.59±0.33c

[0131] 4. Screening of protective agents.

[0132] Each protective agent and mother powder were mixed at a ratio of 1:50, then ultrafinely ground and sieved (200 mesh). Three replicates were set for each sample, and each treatment was irradiated for 40 minutes at a distance of 40 cm from a UV lamp (254 nm, 20 W). The samples were diluted and applied, and the bacterial growth was observed. UV protective agents were screened by comparing the physical and chemical properties of wettable powders before and after UV irradiation, using samples without protective agent and UV treatment as controls.

[0133] Table 6 shows that after UV irradiation, the viable bacterial count was highest when soluble starch was used as the protective agent, reaching 55.7 billion / g. Xanthan gum and soluble starch were used as protective agents, resulting in the highest suspension rate of 79.67%, and a wetting time of 43.33 seconds. The viable bacterial count is a crucial metric in the selection of protective agents. Based on comprehensive considerations, soluble starch was selected as the optimal protective agent.

[0134] Table 6: Screening of protective agents

[0135] Protective agent Number of viable bacteria / (100 million / g) Suspension rate / % Wetting time / s Xanthan gum 527.00±0.07bc 79.67±0.24a 28.27±0.15d dextrin 407.00±0.07d 66.10±0.59b 16.03±0.09e Kaolin 540.00±0.12b 63.17±0.44c 69.87±0.19b Soluble starch 557.00±0.04a 79.67±0.17a 43.33±0.89c CK 507.00±0.07c 57.00±0.58d 79.13±0.19a

[0136] 5. Development of microbial agent formula.

[0137] The best carrier, dispersant, wetting agent and protective agent screened were set to four factors and three levels L9(3 4) design to conduct an orthogonal experiment, mix various adjuvants in proportion to make wettable powders, and determine the optimal formula of microbial agents, i.e. wettable powders, by measuring the suspension rate.

[0138] Diatomaceous earth, polyethylene glycol, sodium carboxymethyl cellulose, and soluble starch were identified as the optimal carriers and adjuvants for wettable powders through single-factor experiments. An orthogonal experiment was conducted using these four materials as screening factors, with three mass fractions set. Using suspension rate as an indicator, the range analysis in Table 7 shows that the four factors, combined with the R values ​​(factor ranges), rank in order: R3 > R2 > R4 > R1. The order of influence of the four factors is: C sodium carboxymethyl cellulose > B polyethylene glycol > D soluble starch > A diatomaceous earth. Therefore, the optimal wettable powder formulation is A2 B3 C2 D3, namely 35% diatomaceous earth, 8% polyethylene glycol, 12% sodium carboxymethyl cellulose, and 0.8% soluble starch.

[0139] Table 7: Orthogonal test results

[0140]

[0141]

[0142] 6. Determination of quality indicators of microbial agents.

[0143] The microbial agent in this embodiment is a wettable powder. The wettable powder is prepared according to the optimal formula, and the viable bacteria count, wetting time, suspension rate, pH value, and fineness are measured.

[0144] According to the screening test of XM18-5 strain wettable powder, the optimal composition was determined to be 35% diatomaceous earth, 8% polyethylene glycol, 12% sodium carboxymethyl cellulose and 0.8% soluble starch. The bacterial powder was added to 100% to make wettable powder. The viable bacterial count of the wettable powder made by this process was 5.58×10 10 cfu / g, suspension rate was 87.50%, wetting time was 88.5s, pH value was 7.04, and fineness was 95.14%. Therefore, it can be seen that all indicators meet the national standards for pesticide wettable powders, indicating that the prepared wettable powder is qualified.

[0145] Example 4

[0146] This embodiment provides a method for preparing microbial granules.

[0147] The 5.58×10 10 cfu / g of XM18-5 wettable powder, adding adhesive, wrapped in amino acid granules to make XM18-5 granules, such as Figure 9 As shown, it is the wettable powder in this embodiment, such as Figure 10 As shown, the microbial granules prepared in this example.

[0148] Example 5

[0149] This example illustrates the use of microbial agents in promoting potato growth.

[0150] First, the test location and materials in this embodiment are described:

[0151] Test Location: The test was conducted in 2023 in Xiji County, Ningxia. Located at the western foot of Liupan Mountain, Xiji County belongs to the arid hilly region of the Loess Plateau and has a typical temperate continental climate with four distinct seasons. The soil type at the test site is black loam soil with medium soil fertility, a pH of 8.45, 6.1 g / kg organic matter, 0.14 g / kg total salt, 0.70 g / kg total nitrogen, 68.5 mg / kg alkaline-hydrolyzable nitrogen, 4.7 mg / kg available nitrogen, and 100 mg / kg available potassium.

[0152] Test variety: Qingshu No. 9. Provided by Xiji County Potato Industry Service Center.

[0153] Test microbial agent: the microbial agent obtained by the proportion in Example 3, named XM18-5 microbial agent in this example.

[0154] Planting method: double-row planting, row spacing 90 cm, plant spacing 35 cm, the cultivation management and field management conditions in the experimental area are exactly the same.

[0155] The experiment included two treatments and one CK. The specific fertilization method is shown in Table 1. Each treatment covered an area of ​​1 mu.

[0156] Treatment 1: XM18-5 microbial agent (2.5 kg per mu, applied in furrows); Treatment 2: Zhongbao Pink Diamond microbial agent (2.5 kg per mu, applied in furrows); Treatment 3: blank control.

[0157] 1. Determination of germination rate.

[0158] The number of seeds sown per plot was counted at the time of sowing, and the number of seedlings emerged per plot was counted 50 days after sowing to calculate the emergence rate: emergence rate (%) = (number of seedlings emerged / number of seeds sown) × 100.

[0159] As shown in Table 8, the potato germination rate of treatment 1 with 80 kg / mu of XM18-5 was 98.78%, which was significantly higher than that of treatment 2 with 40 kg / mu of microbial agent and the control; compared with the control, the average germination rate of treatment 1 increased by 6.13%.

[0160] Table 8: Potato field emergence rate under different treatments

[0161] deal with Average germination rate (%) Ratio CK (±) Process 1 98.78a 6.13％ Process 2 94.16b 1.51％ CK 92.65b /

[0162] 2. Determination of growth indicators.

[0163] During the flowering period, plant height (from stem base to growth point), stem diameter (from main stem base), and root system (root length, root biomass, etc.) were measured using a tape measure and vernier caliper. Ten plants were randomly selected from each plot, and the average value of three replicates was taken.

[0164] Depend on Figures 11 and 12 Significant differences in aboveground and belowground potato biomass were observed between treatments. Results showed that on June 20th, root systems in Treatments 1 and 2 were significantly more developed than in the control, with weight increases of 243.4% and 68.7%, respectively. Aboveground biomass in Treatments 1 and 2 increased by 140.7% and 130%, respectively, compared to the control. Results measured on July 19th were consistent with those on June 20th. Aboveground biomass in Treatments 1 and 2 was significantly greater than in the control, with Treatment 1 showing the greatest increase. Treatment 1 showed the most significant accumulation of aboveground and belowground biomass, indicating that XM18-5 significantly promoted potato growth, boosting both aboveground vegetative growth and root development. See Table 9 for details.

[0165] Table 9: Aboveground and belowground biomass of each treatment at different periods

[0166]

[0167] In terms of plant height, Table 10, Figure 13 The results show that the different treatments had a significant impact on potato plant height. The plant heights of treatments 1 and 2 were significantly higher than those of the control. On July 19, the plant heights increased by 35.7 cm and 9 cm, respectively, compared to the control.

[0168] Table 10: Plant height of each treatment at different periods

[0169]

[0170] In terms of root length, Table 11, Figure 14 The results show that the different treatments had a significant impact on potato root length. Plant heights in treatments 1 and 2 were significantly higher than those in the control. On July 19, the root lengths of the plants increased by 19.2 cm and 9 cm, respectively, compared to the control.

[0171] Table 11: Root length of each treatment at different periods

[0172]

[0173] Stem thickness, Figure 15The results show that the different treatments had a significant impact on potato stem diameter. The plant heights in Treatments 1 and 2 were significantly higher than those in the control. On July 19, the stem diameters of potatoes increased by 5.8 mm and 2.2 mm, respectively, compared to the control.

[0174] comprehensive Figure 13 、 Figure 14 、 Figure 15 Analysis revealed that the effects of the XM18-5 microbial inoculant on potato plant height, stem diameter, and root length were significantly greater than those of the control treatment. Significant differences were observed in plant height, stem diameter, and root length between Treatment 1, Treatment 2, and the control. This suggests that XM18-5 significantly promotes potato growth and root development. Potato seedlings exhibited no deformities or plant dieback, demonstrating that the microbial inoculant is safe for potato growth and suitable for widespread use in production.

[0175] Example 6

[0176] The test location and materials in this embodiment are the same as those in Example 5. This embodiment describes the test results of soil index determination.

[0177] Before fertilization and during harvest, 1 kg of soil was collected from the field control and demonstration areas to measure soil organic matter, total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus, available potassium, effective potassium, pH value, and cations.

[0178] Application of XM18-5 to potato fields altered soil physical and chemical properties. Table 12 shows that 84 days after application, soil hydrolyzable nitrogen, available phosphorus, and available potassium significantly decreased in Treatments 1 and 2 compared to pre-application levels. Hydrolyzable nitrogen decreased by -50.4%, -48.6%, and -17.1% in Treatments 1, 2, and CK, respectively. Available phosphorus decreased by -36.6%, -36.7%, and +36.2% in Treatments 1, 2, and CK, respectively. Available potassium decreased by -18.5%, -32.2%, and -3.7% in Treatments 1, 2, and CK, respectively. This downward trend was more pronounced than in the CK treatment, indicating that the application of XM18-5 to the soil decomposed the amino acids and organic matter in the microbial agent to produce humus, which has a strong ion adsorption effect, regulating soil anions and cations, thereby promoting the utilization and absorption of nitrogen, phosphorus, and potassium from the soil and fertilizer by potatoes.

[0179] Table 12: Test results of soil indicators before and after application of microbial agents

[0180]

[0181] Example 7

[0182] The test location and materials in this example are the same as those in Example 5. This example illustrates the application of microbial agents in potato disease prevention.

[0183] 1. Effective in preventing early blight of potato.

[0184] The disease base will be surveyed in early July 2023, the disease index will be surveyed for the first time in early August, and the disease index will be surveyed for the second time in late August. The interval between the first and second surveys will be 7-10 days, for a total of three surveys.

[0185] Random sampling was conducted at five points in each plot, with 2 to 3 plants surveyed at each point. Ten leaves of each plant were surveyed at the upper, middle and lower parts, and the plants were graded based on the percentage of the lesion area on each leaf to the total leaf area.

[0186] Grading method:

[0187] Level 0: no lesions; Level 1: the area of ​​lesions accounts for less than 5% of the total leaf area; Level 3: the area of ​​lesions accounts for 6% to 10% of the total leaf area; Level 5: the area of ​​lesions accounts for 11% to 20% of the total leaf area; Level 7: the area of ​​lesions accounts for 21% to 50% of the total leaf area; Level 9: the area of ​​lesions accounts for more than 50% of the total leaf area.

[0188] The prevention effect is calculated according to formula (1) and formula (2):

[0189]

[0190] Where: CK0 is the disease index of the blank control area before application; CK1 is the disease index of the blank control area after application; PT0 is the disease index of the chemical treatment area before application; PT1 is the disease index of the chemical treatment area after application. If the disease base is not investigated before application, the control effect is calculated according to formula (3):

[0191]

[0192] According to the standardized data analysis procedure, the control effect was analyzed by Duncan's new multiple range (DMRT) method using statistical software such as EXCEL and DPS, and the results were analyzed and discussed.

[0193] Table 13 shows the disease index of potato early blight and the control efficacy of each treatment. On July 4, no significant differences were found among the treatments. On July 19, the disease index of potato early blight in each experimental plot changed, and the control efficacy varied to varying degrees. The blank control had the highest disease index of 8.78, while the disease indexes for Treatments 1 and 2 were 2.99 and 3.45, respectively, with control efficacy rates of 68.68% and 60.71%, respectively. On August 24, the disease index of potato early blight in each experimental plot increased significantly, and there were significant differences in control efficacy among the treatments. The disease index of the control was significantly higher than all treatments (P < 0.05), reaching 18.75. The disease indexes for Treatments 1 and 2 were 2.99 and 3.45, respectively, with control efficacy rates of 64.53% and 45.33%, respectively.

[0194] Table 13: Field control effects on potato early blight

[0195]

[0196] 2. Effective in preventing potato late blight

[0197] The disease base will be surveyed in mid-to-late July 2023, the disease index will be surveyed for the first time in mid-to-late August, and the disease index will be surveyed for the second time at the end of August. There will be an interval of 7 to 10 days between the first and second surveys, for a total of three surveys.

[0198] Samples were collected at five diagonal points in each plot, with three plants surveyed at each point, for a total of 15 plants. Ten compound leaves were collected from each plant, for a total of 150 leaves. Each leaf was graded and recorded based on the percentage of lesions to leaf area. Disease index and control efficacy were calculated, and a new multiple range significance analysis was performed on control efficacy. The average of four replicates was used for each treatment.

[0199] Potato late blight grading standards:

[0200] Level 0: no lesions; Level 1: the area of ​​lesions accounts for less than 5% of the entire leaf area; Level 3: the area of ​​lesions accounts for 6% to 10% of the entire leaf area; Level 5: the area of ​​lesions accounts for 11% to 25% of the entire leaf area; Level 7: the area of ​​lesions accounts for 26% to 50% of the entire leaf area; Level 9: the area of ​​lesions accounts for more than 50% of the entire leaf area.

[0201] Disease index = Σ(number of diseased leaves at each level × relative level value) × 100 / (total number of leaves surveyed × 9);

[0202] Control effect (%) = [1-(disease index of blank control area before application × disease index of treated area after application) / (disease index of blank control area after application × disease index of treated area before application)] × 100;

[0203] Observe whether there is any pesticide damage during the potato growth period 7 to 14 days after application. If pesticide damage occurs, investigate and record it according to the pesticide damage classification method.

[0204] The prevention effect is calculated according to formula (4) and formula (5):

[0205]

[0206] Where: CK0 is the disease index of the blank control area before application; CK1 is the disease index of the blank control area after application; PT0 is the disease index of the chemical treatment area before application; PT1 is the disease index of the chemical treatment area after application. If the disease base is not investigated before application, the control effect is calculated according to formula (6):

[0207]

[0208] According to the standardized data analysis procedure, the control effect was analyzed by Duncan's new multiple range (DMRT) method using statistical software such as EXCEL and DPS, and the results were analyzed and discussed.

[0209] Table 14 shows the disease index of potato late blight and the control efficacy of each treatment. On July 19, the disease base number for each treatment showed no significant difference. On August 8, the disease index for the control treatment was 31.5, significantly higher than the other two treatments. Treatment 1 achieved a 63.68% control efficacy against potato late blight, while Treatment 2 achieved a 51.65% efficacy. On August 24, the disease index for the control area was 55.25, significantly higher than Treatment 1's 26.55 and Treatment 2's 37.9. The control efficacy of Treatments 1 and 2 was 51.95% and 31.29%, respectively. Analysis of variance revealed a significant difference in efficacy between Treatments 1 and 2.

[0210] Table 14: Field control effect of microbial agent treatment on potato late blight

[0211]

[0212] 3. Effective in preventing potato scab.

[0213] The potato scab disease grading standards are:

[0214] Level 0, no lesions; Level 1, the area of ​​lesions accounts for less than 1% of the total leaf area; Level 3, the area of ​​lesions accounts for 1% to 10% of the total leaf area; Level 5, the area of ​​lesions accounts for 11% to 20% of the total leaf area; Level 7, the area of ​​lesions accounts for 20% to 50% of the total leaf area; Level 9, the area of ​​lesions accounts for more than 50% of the total leaf area.

[0215] Calculation formula for disease index and relative disease resistance index:

[0216] Disease index = Σ(number of diseased potatoes at each level × relative level value) × 100 / (total number of surveyed potatoes × 9);

[0217] Control effect (%) = [1-(disease index of blank control area before application × disease index of treated area after application) / (\(disease index of blank control area after application × disease index of treated area before application)] × 100.

[0218] From Table 15, it can be seen that XM18-5 has a good control effect on potato scab. The disease index of treatment 1 is the lowest, which is 2.95, and the control effect is 71.81%. The second is treatment 2, with a disease index of 6.54 and a control effect of 36.87%.

[0219] Table 15: Field control effect of microbial agent treatment on potato scab

[0220] deal with Disease index Control effect (%) Process 1 2.95c 71.81a Process 2 6.54b 36.87b CK 10.36a /

[0221] Example 8

[0222] The test site and materials in this example are the same as those in Example 5. This example illustrates the determination of the number of microorganisms in the potato rhizosphere soil.

[0223] The number of fungi, bacteria, and actinomycetes in the soil was determined by the dilution plate method. Martin's Bengal rose medium was used for fungi, beef extract peptone medium was used for bacteria, and modified Gao's medium No. 1 was used for actinomycetes. The results were expressed as the number of microbial colony forming units (CFU·g) per 1 g of fresh soil. -1 )express.

[0224] As shown in Table 16, the number of potato rhizosphere fungi in each treatment showed an initial increase followed by a decrease as the potato growth cycle progressed. The order of fungal abundance in potato soil at different stages was generally CK > Treatment 2 > Treatment 1, with the control containing the highest number of pathogenic fungi. This suggests that applying XM18-5 as a carrier of functional microorganisms in the soil provides a large source of beneficial microorganisms, particularly the colonization of beneficial microorganisms such as Bacillus spp. in the microbial inoculant, effectively reducing the number of harmful fungi in the soil. This reduction in fungal abundance can, in turn, reduce the risk of soil-borne diseases.

[0225] Table 16: Effects of different treatments on the number of fungi in the potato rhizosphere

[0226]

[0227] Table 17 shows that the number of soil bacteria gradually increases as the potato growth cycle progresses. Starting from the seedling stage, the number of bacteria in the potato rhizosphere soil gradually increases, reaching its peak at the harvest stage. Treatment 1 had the highest bacterial count during the seedling stage. During the mature stage, bacterial counts increased across all treatments, with a smaller increase in Treatment 2, and the highest count in Treatment 1. At both the flowering and harvest stages, bacterial counts were higher in the inoculant treatments, with significant differences between treatments. This suggests that after applying XM18-5 to the soil, beneficial bacteria such as Bacillus spp. in the microbial inoculant can colonize the soil in large numbers, antagonizing pathogenic fungi and reducing the incidence of soil-borne diseases. Furthermore, the proliferation of beneficial bacteria promotes the transformation and absorption of soil nutrients.

[0228] Table 17: Effects of different treatments on the number of rhizospheric bacteria in potato

[0229]

[0230] Table 18 shows that the number of soil actinomycetes in all treatments showed an initial upward and then downward trend throughout the potato growth cycle. Starting from the seedling stage, the number of actinomycetes in the rhizosphere soil of each treatment gradually increased, reaching its peak at the mature plant stage, and then slowly declined. The highest number of actinomycetes was observed in Treatment 1, followed by Treatment 2 at the seedling, mature plant, flowering, and harvest stages, with significant differences among the treatments. This suggests that the application of XM18-5 to the soil promotes the growth of actinomycetes, which in turn promote the conversion of soil organic matter and produce antibiotics that have some antagonistic effects on soil-borne diseases, thereby reducing the number of pathogenic fungi in the soil.

[0231] Table 18: Effects of different treatments on the number of actinomycetes in the potato rhizosphere

[0232]

[0233] Example 9

[0234] The test location and materials in this example are the same as those in Example 5. This example illustrates the yield and economic benefits of potatoes.

[0235] Before potato harvest, yield measurements were conducted within each treatment. To eliminate marginal effects, samples were taken from five locations along the middle two ridges. Tubers were categorized as large, small (large ≥ 50 g, small < 50 g), and diseased. Potato yield and commercial yield were calculated.

[0236] The field yield test results are shown in Table 19. There are significant differences among the treatments. The yield of treatment 1 is 2949.33 kg / 667 m 2 , the output of treatment 2 is 2120kg / 667m 2 The control yield was 1376.67kg / 667m2 Furthermore, calculations of the commercial potato yield revealed that treatment 1 had the highest commercial potato yield of 78.95%, treatment 2 had a yield of 63.04%, and the control had a yield of 58.06%, showing significant differences among treatments. In summary, XM18-5 can significantly increase potato yield and the proportion of commercial potatoes.

[0237] Table 19: Effects of different treatments on potato yield and economic benefits

[0238]

[0239]

[0240] The economic benefit analysis showed that, based on the potato market price of 1.6 yuan / kg, the total output value per mu of treatment 1 was 3,967.43 yuan, the total output value per mu of treatment 2 was 2,800.50 yuan, and the total output value per mu of the control treatment was 1,771.17 yuan. Compared with the control, the increased output value per mu of treatment 1 and treatment 2 was 2,196.26 yuan and 1,029.33 yuan, respectively, with significant economic benefits.

[0241] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A microbial agent, characterized in that: The active ingredient of the microbial agent is Bacillus velezensis XM18-5, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC 25698.

2. The microbial agent according to claim 1, characterized in that The microbial agent is composed of the following components in percentage by mass: 30% to 40% of a carrier, 2% to 8% of a wetting agent, 8% to 16% of a dispersant, 0.2% to 0.8% of a protective agent, and 35.2% to 59.8% of bacterial powder. The carrier is one of diatomaceous earth, kaolin, attapulgite, and white carbon black; the wetting agent is one of sodium lauryl sulfate, polyethylene glycol, and calcium chloride; the dispersant is one of sodium lignosulfonate, sodium tripolyphosphate, sodium methylene disulfonate, gum arabic, and sodium carboxymethyl cellulose; and the protective agent is one of sodium humate, xanthan gum, kaolin, dextrin, and soluble starch; The bacterial powder includes Bacillus velezensis XM18-5.

3. The microbial agent according to claim 2, characterized in that The microbial inoculant is composed of the following components in percentage by mass: 35% diatomaceous earth, 8% polyethylene glycol, 12% sodium carboxymethyl cellulose, 0.8% soluble starch, and 35.2% bacterial powder.

4. The microbial agent according to claim 2 or 3, characterized in that Calculated by mass percentage, the preparation steps of the bacterial powder include: Step 1: Seed solution preparation: Step 101: The strain XM18-5 was transferred to PDA medium and activated for 24 hours. A single colony was picked and streaked for subculture, and cultured for two generations until the normal metabolic level was reached. Step 102: Pick a ring of XM18-5 and transfer it to NB medium, culture it at 30°C and 180 rpm for 24 hours to prepare the seed solution for later use; Step 2: Fermentation broth preparation: Step 201: Inoculate the reserved seed solution in step 102 into NB culture medium at an inoculum volume of 5% to 10% (V / V), and culture at 34° C. and 180 rpm for 48 hours; Step 202: Measure the spore rate using a hemocytometer to ensure that the spore rate is ≥90%, and the fermentation liquid is ready for use. Step 3: Centrifuge the fermentation broth at 4°C and 8000 rpm for 10 min, discard the supernatant, and obtain a concentrated bacterial solution; Step 4: Preparation of bacterial powder: Step 401: mixing 60% to 65% corn straw, 20% to 30% bran, 2.5% to 3.0% corn, 1.40% to 1.48% ammonium sulfate, and 0.10% to 0.15% manganese sulfate, adding 6% to 10% of the concentrated bacterial solution obtained in step 3 as an inoculum, and solid-state fermenting in a 37° C. incubator for 48 hours to obtain a fermentation product; Step 402: drying the fermentation product obtained in step 401 in a 45° C. oven and then ultrafine grinding the product to prepare final bacterial powder.

5. A microbial granule, characterized in that: The microbial granules are prepared by adding a binder to the microbial agent according to claim 1.

6. Use of the microbial agent according to claim 1 or the microbial granules according to claim 5 in preventing and treating potato blight.

7. The use according to claim 6, characterized in that The diseases include potato early blight, potato late blight, potato black mole, potato scab and potato dry rot.

8. Use of the microbial agent according to claim 1 or the microbial granules according to claim 5 in improving the emergence rate of potatoes.

9. Use of the microbial agent according to claim 1 or the microbial granules according to claim 5 in promoting potato growth.

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